DNA topoisomerases are enzymes that control the topological state of DNA in all cells; they have central roles in DNA replication and transcription. They are classified into two types, I and II, depending on whether they catalyze reactions involving the breakage of one or both strands of DNA. Structural and mechanistic distinctions have led to further classifications: IA, IB, IC, IIA, and IIB. The essence of the topoisomerase reaction is the ability of the enzymes to stabilize transient breaks in DNA, via the formation of tyrosyl-phosphate covalent intermediates. The essential nature of topoisomerases and their ability to stabilize DNA breaks has led to them being key targets for antibacterial and anticancer agents. This chapter reviews the basic features of topoisomerases focussing mainly on the prokaryotic enzymes. We highlight recent structural advances that have given new insight into topoisomerase mechanisms and into the molecular basis of the action of topoisomerase-specific drugs.

118.Morrison A, Cozzarelli NR. 1981. Contacts between DNA gyrase and its binding site on DNA: features of symmetry and asymmetry revealed by protection from nucleases. Proc Natl Acad Sci USA78:1416–1420. [PubMed][CrossRef]

193.Willmott CJR, Maxwell A. 1993. A single point mutation in the DNA gyrase A protein greatly reduces the binding of fluoroquinolones to the gyrase-DNA complex. Antimicrob Agents Chemother37:126–127.[PubMed]

DNA topoisomerases are enzymes that control the topological state of DNA in all cells; they have central roles in DNA replication and transcription. They are classified into two types, I and II, depending on whether they catalyze reactions involving the breakage of one or both strands of DNA. Structural and mechanistic distinctions have led to further classifications: IA, IB, IC, IIA, and IIB. The essence of the topoisomerase reaction is the ability of the enzymes to stabilize transient breaks in DNA, via the formation of tyrosyl-phosphate covalent intermediates. The essential nature of topoisomerases and their ability to stabilize DNA breaks has led to them being key targets for antibacterial and anticancer agents. This chapter reviews the basic features of topoisomerases focussing mainly on the prokaryotic enzymes. We highlight recent structural advances that have given new insight into topoisomerase mechanisms and into the molecular basis of the action of topoisomerase-specific drugs.

Model of the topology of a replicating chromosome. The chromosome is separated into domains, with the boundaries represented as orange boxes; the replication fork is in the center. Positive supercoiling occurs ahead of the replication fork, and precatenanes may form behind it.

Reprinted from the Proceedings of the National Academy of Sciences of the United States of America (8) with the permission of the publisher.

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Figure 3

Model of the topology of a replicating chromosome. The chromosome is separated into domains, with the boundaries represented as orange boxes; the replication fork is in the center. Positive supercoiling occurs ahead of the replication fork, and precatenanes may form behind it.

Reprinted from the Proceedings of the National Academy of Sciences of the United States of America (8) with the permission of the publisher.

Formation of catenated DNA at the termination of replication. (a and b) Converging replication forks (a) lead to the interwinding of daughter molecules and the formation of precatenanes (b). (c) Upon the completion of replication, the products are catenated DNA circles.

Reprinted from DNA Topology (11) with the permission of the publisher.

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Figure 4

Formation of catenated DNA at the termination of replication. (a and b) Converging replication forks (a) lead to the interwinding of daughter molecules and the formation of precatenanes (b). (c) Upon the completion of replication, the products are catenated DNA circles.

Reprinted from DNA Topology (11) with the permission of the publisher.

Alignment of the domains of the type II topoisomerases based on amino acid sequence homologies to E. coli gyrase. (a) Type IIA topoisomerases. (b) Topo VI, the only topoisomerase in the type IIB class.

Reprinted from the Annual Review of Biochemistry (2) with the permission of the publisher.

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Figure 6

Alignment of the domains of the type II topoisomerases based on amino acid sequence homologies to E. coli gyrase. (a) Type IIA topoisomerases. (b) Topo VI, the only topoisomerase in the type IIB class.

Reprinted from the Annual Review of Biochemistry (2) with the permission of the publisher.

Proposed mechanism for E. coli topo I. The enzyme binds DNA and cleaves one strand, forming a 5′-phosphodiester linkage (black circle). The complementary strand is passed through the gap and into the central cavity of the enzyme. The nick is resealed, and the strand is released.

Reprinted from DNA Topology (11) with the permission of the publisher.

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Figure 8

Proposed mechanism for E. coli topo I. The enzyme binds DNA and cleaves one strand, forming a 5′-phosphodiester linkage (black circle). The complementary strand is passed through the gap and into the central cavity of the enzyme. The nick is resealed, and the strand is released.

Reprinted from DNA Topology (11) with the permission of the publisher.

The 43-kDa N-terminal domain of E. coli ParE, with one monomer shown in blue and the other in yellow. ADPNP is depicted in stick form near the topoisomerase of the molecule. Reprinted from Antimicrobial Agents and Chemotherapy (83).

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Figure 10

The 43-kDa N-terminal domain of E. coli ParE, with one monomer shown in blue and the other in yellow. ADPNP is depicted in stick form near the topoisomerase of the molecule. Reprinted from Antimicrobial Agents and Chemotherapy (83).

Domain structure of the DNA gyrase subunits. GyrA consists of an N-terminal 59-kDa domain and a C-terminal 35-kDa domain. The QRDR and active-site Tyr122 are marked. The GyrB subunit consists of an N-terminal 43-kDa domain and a C-terminal 47-kDa domain.

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Figure 12

Domain structure of the DNA gyrase subunits. GyrA consists of an N-terminal 59-kDa domain and a C-terminal 35-kDa domain. The QRDR and active-site Tyr122 are marked. The GyrB subunit consists of an N-terminal 43-kDa domain and a C-terminal 47-kDa domain.

Structure of the 35-kDa C-terminal domain of GyrA from B. burgdorferi. (a) The protein structure is shown as ribbons, with each of the six blades being a different color. (b) Representation of the β-pinwheel. One strand is highlighted in red.

Adapted from the Proceedings of the National Academy of Sciences of the United States of America (87) with the permission of the publisher.

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Figure 15

Structure of the 35-kDa C-terminal domain of GyrA from B. burgdorferi. (a) The protein structure is shown as ribbons, with each of the six blades being a different color. (b) Representation of the β-pinwheel. One strand is highlighted in red.

Adapted from the Proceedings of the National Academy of Sciences of the United States of America (87) with the permission of the publisher.

Solution structures of GyrA (a) and GyrB (b) as determined by SAXS and ab initio modeling (88, 105). (a) The GyrA59 structure (86) is shown in blue, with the active-site tyrosines shown in yellow as space-filling representations. The GyrA59 carboxy termini are colored green. A model of the C-terminal domain (87) is shown as orange ribbons and is attached to GyrA59 by a linker. (b) The model of GyrB is shown in grey. The GyrB43 structure (104) is shown as blue ribbons. The TOPRIM region (57), tail 1, and tail 2 are shown as red, purple, and green ribbons, respectively.

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Figure 16

Solution structures of GyrA (a) and GyrB (b) as determined by SAXS and ab initio modeling (88, 105). (a) The GyrA59 structure (86) is shown in blue, with the active-site tyrosines shown in yellow as space-filling representations. The GyrA59 carboxy termini are colored green. A model of the C-terminal domain (87) is shown as orange ribbons and is attached to GyrA59 by a linker. (b) The model of GyrB is shown in grey. The GyrB43 structure (104) is shown as blue ribbons. The TOPRIM region (57), tail 1, and tail 2 are shown as red, purple, and green ribbons, respectively.

Model for negative supercoiling by DNA gyrase. The domains are colored as follows: GyrB N-terminal domain, yellow; GyrB C-terminal domain, orange; GyrA N-terminal domain, dark blue; and GyrA C-terminal domain, light blue. The G and T DNA segments are colored green and red, respectively. (1 and 2) The G segment binds across GyrA, and the GyrA C-terminal domain wraps the DNA to present the T segment. (2 and 3) ATP is bound, which closes the GyrB clamp. (3 and 4) The G segment is cleaved, and the T segment passes through. (4 and 5) The T segment exits through the bottom gate, and the G segment is religated. (5 and 2) The hydrolysis of ATP resets the enzyme.

Reprinted from Biochemistry (129) with the permission of the publisher.

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Figure 17

Model for negative supercoiling by DNA gyrase. The domains are colored as follows: GyrB N-terminal domain, yellow; GyrB C-terminal domain, orange; GyrA N-terminal domain, dark blue; and GyrA C-terminal domain, light blue. The G and T DNA segments are colored green and red, respectively. (1 and 2) The G segment binds across GyrA, and the GyrA C-terminal domain wraps the DNA to present the T segment. (2 and 3) ATP is bound, which closes the GyrB clamp. (3 and 4) The G segment is cleaved, and the T segment passes through. (4 and 5) The T segment exits through the bottom gate, and the G segment is religated. (5 and 2) The hydrolysis of ATP resets the enzyme.

Reprinted from Biochemistry (129) with the permission of the publisher.

Chemical structures of a selection of quinolones. Nalidixic acid and oxolinic acid are examples of older acidic quinolones. Ciprofloxacin and norfloxacin are examples of the amphoteric fluoroquinolones (expanded-spectrum compounds).

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Figure 20

Chemical structures of a selection of quinolones. Nalidixic acid and oxolinic acid are examples of older acidic quinolones. Ciprofloxacin and norfloxacin are examples of the amphoteric fluoroquinolones (expanded-spectrum compounds).